We have developed chromosomal cross-link analysis (CXLA) technology for the rapid purification of proteins covalently attached to DNA. CXLA produces proteins in amounts and purity sufficient for unambiguous identification of the proteins and their post-translational modifications by proteomic methods. We will use CXLA together with biochemical and molecular genetic methods to test several hypotheses and models concerning the structure and functions of post-translational modifications of topoisomerases that result from their targeting by anticancer drugs and treatments. Specific Aim I will test the hypothesis that sumoylation of topoisomerase I is involved in detection, signaling and/or processing of topoisomerase I mediated DNA damage by camptothecins. Models for the specific function of sumoylation will be tested and alternate hypotheses will be tested if necessary. Specific Aim II will test the hypothesis that ubiquitin family modifications of topoisomerase II and their function vary depending on the isozyme, its function and the anticancer drugs stabilizing topo II-DNA cleavage complexes. Specific Aim III will test several hypotheses for functions of topoisomerase phosphorylation: (1), That phosphorylation at specific sites is required for DNA cleavage (2), that ubiquitination and sumoylation are driven by phosphorylation at specific sites (3), that prolyl isomerization is required for sumoylation of topoisomerases in drug-stabilized DNA cleavage complexes. Specific Aim IV will test the hypotheses that (1) repair enzymes known from lower phyla, but not yet discovered in humans, are present at sites of topoisomerase mediated DNA damage (2) that different sets of repair enzymes are recruited to replication forks damaged by topo I poisons, topo II poisons and hydroxyurea as a result of differences in fork damage caused by these agents. Studies under Aim IV are also expected to yield new information on the sequence and kinetics of recruitment of repair enzymes to damaged forks, and information on direct interactions of repair proteins and steps in fork repair.